Hfq proteins primarily function as chaperones in the bacterial cell, facilitating the interaction between small RNAs (sRNAs) and their target mRNAs to regulate gene expression. The role of Hfq and its associated sRNA regulation has not been fully or clearly elucidated in Gram-positive bacteria, despite the expression of numerous sRNAs in these species. Unlike most bacteria, Bacillus anthracis possesses multiple copies of hfq within its genome. Two copies are located on the B. anthracis chromosome (genes for Hfq1 and Hfq2), and the third (gene for Hfq3) is encoded by the pXO1 virulence plasmid. In the first project executed in 2017, we examined the effects of overexpressing Hfq3 in B. anthracis as a means of identifying potential Hfq3-regulated pathways. We selected Hfq3 for further analysis due to its sequence divergence from other Bacillus Hfq proteins, as well as its ability to form hexamers and its presence on one of the virulence plasmids of B. anthracis. We observed a severe growth defect associated with the overexpression of Hfq3 in B. anthracis, but not in B. subtilis. We made many mutations on the two surfaces of the Hfq hexamer and on its rim, targeting residues known in the Hfqs of other bacterial to have specific roles. Based on this mutational analysis, we determined that the growth-inhibitory phenotype is dependent on residues on the distal face of Hfq3 that are associated in other species with mRNA binding. Thus, we have identified a novel phenotype associated with Hfq overexpression in a Gram-positive species, contributing to our current understanding of structure-function relationships of Hfq proteins in Gram-positive bacteria. In 2017, we also continued to use tyrosine site-specific recombinases (T-SSR) to perform genetic modifications in B. anthracis. The systems we developed are Cre-loxP, Flp/FRT, and IntXO-PSL. These rhree distinct tyrosine recombinase systems were used for creation of a B. anthracis sporulation-deficient, plasmid-free strain deleted for ten proteases which had been identified by proteomic analysis as being present in the B. anthracis secretome. This strain was used successfully for production of various recombinant proteins, including several that are candidates for inclusion in improved anthrax vaccines. The expression of the key virulence factors of B. anthracis are tightly regulated, responding to the presence of carbon dioxide, which is a signal that the bacteria are within a mammalian host. A key regulator of the response to carbon dioxide is the AtxA protein. We have previously characterized the function of AtxA using Next Generation sequencing. In a third project done during 2017, we extended study of AtxA by constructing multi-protease deficient B. anthracis strains having chromosomal insertions of 1, 2, or 3 adjacent atxA genes. We found a positive correlation between the atxA copy number and the expression level of the pagA gene encoding B. anthracis protective antigen, when strains were grown in a carbon dioxide atmosphere. In addition to providing information about the normal role of AtxA, this work has produced plasmids that support higher levels of expression of heterologous proteins in multi-protease deficient B. anthracis strains.